Cytokine-mediated activation of inducible nitric oxide synthase (iNOS) in monocytes or macrophages is species specific. In contrast to rat or mouse, human macrophages do not produce measurable levels of nitric oxide (NO) when induced by inflammatory mediators. Exposure to non-cytokine mediators such as tumor cells or viruses, however, has recently been shown to activate human iNOS. NO production in response to these mediators is much lower than that seen for rat or mouse cells and often requires several days of stimulation. We have found that the synthetic, double- stranded polyribonucleotide polyinosinic-polycytidilic acid (Poly I:C), commonly used to mimic viral exposure, activated iNOS in human monocyte- derived macrophages (MDM). The production of NO, measured by nitrite accumulation, was detected after 24 hours of stimulation with Poly I:C. The single-stranded polyribonucleotide Poly I, but not Poly C, also increased NO production. Nitrite production was enhanced when the MDM were primed (pretreated) with gamma- or alpha-interferon or other immune mediators such as IL-4 and was reduced by the iNOS inhibitor, N-methyl-L- arginine(L-MMMA). The use of Poly I:C to initiate NO production in human macrophages provides a useful tool to study the differences between the commonly used animal models and human cells and may provide insight into the pathophysiological significance of these differences.